Numerous studies show that alcohol (ethanol) consumption affects neuronal signaling systems, resulting in behavioral disorders, decline of cognitive functions, brain atrophy, and, in chronic alcohol users dementia. Alcohol use and/or abuse can also adversely affect the course of a variety of neurobehavioral diseases, inflammation disorders and enhances susceptibility to bacterial infections, and HIV-related neurological disease. For alcohol to exert its action inside the brain and cause neuronal damage, it must pass the blood-brain barrier (BBB). During this process alcohol affects the metabolism and function of the BBB. The effects of alcohol on the BBB are unclear and one of the least explored areas of alcohol research. To study BBB function in alcohol use, we have used a unique in-vitro model of the human BBB. We tested alcohol in various concentrations, such as found in moderate alcohol users, acute "binge" drinkers, and in chronic alcoholics and found that alcohol increased BBB permeability. Moderate to higher alcohol displayed an immediate effect, whereas lower concentrations affected BBB after longer duration of treatment. Our data show that alcohol-mediated BBB dysfunction involves a rearrangement of the cytoskeleton and is mediated through NADPH-dependent reactive oxygen species (ROS). Activation of the BBB and increased BBB permeability may result in a flux of plasma factors across the BBB. The influx of plasma factors may lead to exacerbation of the direct and detrimental effects of alcohol on astrocyte and neuronal functioning. We hypothesize that alcohol deregulates endothelial signaling mechanisms that control barrier integrity through NADPH-dependent ROS mediated mechanisms. Blocking alcohol-mediated BBB dysfunction may significantly limit the negative effects of alcohol use on the brain parenchyma, including neuronal functioning. In this exploratory study, we propose to further investigate the kinetics and underlying intracellular signaling mechanism(s) of alcohol mediated BBB dysfunction using an in vitro model of the human BBB and extend this to an in-vivo mouse model for alcohol abuse. It is anticipated that these exploratory studies will provide information on the time- and dose-dependent relationship whereby alcohol affects the BBB, the underlying signaling mechanisms and establish a good in vivo model for alcohol-BBB dysfunction. The long term goal is to further develop a program in alcohol mediated BBB dysfunction and development of BBB protective drugs. This resubmitted exploratory R21 will address a novel line of investigation. Public Health Relevance: Drinking of alcoholic beverages affects the brain, resulting in behavioral disorders, affects cognitive functions and can even lead to dementia. We found that alcohol negatively affected properties of the blood vessels in the brain, the so called blood brain barrier (BBB), leading to an increased permeability to small solutes and this may worsen the effects of alcohol on the neuronal and glial cells inside the brain. In this exploratory study, we propose to use and in vitro model of the human BBB and an in vivo mouse model for alcohol use to further investigate the mechanisms whereby alcohol affects the BBB.